Double immunofluorescence microscopic technique for accurate differentiation of extracellularly and intracellularly located bacteria in cell culture

Abstract

A double immunofluorescence staining technique is described for differentiation between cell-attached (extracellular) and ingested (intracellular) bacteria by HEp-2 cells in cell culture monolayers. This method is based upon the observation that membranes of viable mammalian cells are impermeable for antibodies but are rendered permeable by treatment with fixatives. Consequently, extracellular bacteria can be stained by specific rhodamine-labeled antibodies before fixation, and intracellular bacteria can be visualized by treatment with specific fluorescein-labeled antibodies after fixation. The accuracy and simplicity of this method is demonstrated with HEp-2 cell culture monolayers as target cells and an isogenic pair of Yersinia enterocolitica, one of which is phagocytosis resistant and the other of which is phagocytosis sensitive. Furthermore, it is shown that this staining technique is also applicable for studying the interaction of bacteria with macrophages and fibroblasts.

Post‐translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA levels and susceptibility to Yersinia invasion

“…These differences could not be accounted for by changes in cell viability or cell growth given the controls and time frame of the experiment. To confirm the initial observation by an alternate method, we used a double fluorescence staining technique that enables visualization of extracellular and intracellular bacteria in the same cell [25]. The results concurred with the data from the gentamicin-based invasion assay.…”

“…Double staining of intra-and extracellular Yersinia was performed essentially as described previously [25]. HEK293T cells were seeded in a 12-well plate (2 · 10 5 well )1 ) on coverslips and grown for 12 h in DMEM supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin ⁄ streptomycin.…”

Distinct mechanisms of integrin binding by Yersinia pseudotuberculosis adhesins determine the phagocytic response of host macrophages

“…J774A.1 macrophages were infected with these strains at a multiplicity of infection (moi) of 20:1 for 1 h to investigate the functional relationship of invasin and YadA during phagocytosis. Unbound bacteria were removed by gentle washing and the cells were fixed and stained using a double-label immunofluorescence technique to quantify the number of attached and internalized bacteria (Heesemann and Laufs, 1985;Black and Bliska, 1997;Weidow et al ., 2000;Bruce-Staskal et al ., 2002). In this assay, bacteria were stained before cell permeabilization with a fluoroscein isothiocyanate (FITC)-conjugated antibody, and then a second time following permeabilization with a Texas red (TR)-conjugated antibody.…”

“…Cells were infected at 37∞C in 7.5% CO 2 for 60 min in 1 ml of binding buffer containing the designated Y. pseudotuberculosis strains at an moi of approximately 20:1. Cells were fixed and stained as previously described (Heesemann and Laufs, 1985;Bruce-Staskal et al, 2002). All antibodies were diluted in PBS containing 2% BSA, and all incubations were at room temperature for 30 min.…”

Epithelial invasion and cell lysis by virulent strains of Streptococcus suis is enhanced by the presence of suilysin

“…Using a di¡erential £uorescent labelling technique [17], whereby external, adherent bacteria were stained red and green and internal bacteria were stained only green, the adherence of di¡erent strains of bacteria to the HEp-2 epithelial cells as well as the internalisation of bacteria were quanti¢ed. Fig.…”